Random insertion and deletion mutagenesis for construction of protein library containing nonnatural amino acids

Incorporation of nonnatural amino acids into proteins.

The genetic code is established by the aminoacylation of transfer RNA, reactions in which each amino acid is linked to its cognate tRNA that, in turn, harbors the nucleotide triplet (anticodon) specific to the amino acid. The accuracy of aminoacylation is essential for building and maintaining the universal tree of life. The ability to manipulate and expand the code holds promise for the develo...

Pentapeptide scanning mutagenesis: random insertion of a variable five amino acid cassette in a target protein.

A new insertion method for probing protein functional organization was developed. The method relies on the random insertion of transposon Tn 4430 and subsequent in vitro deletion of the bulk of the transposon after which a 15 bp insertion remains within the target gene. This results in pentapeptide insertions randomly distributed in the target protein. Characterization of 23 pentapeptide insert...

Versatile PCR-mediated insertion or deletion mutagenesis.

Insertion and deletion mutagenesis of the human cytomegalovirus genome.

Studies on human cytomegalovirus (CMV) have been limited by a paucity of molecular genetic techniques available for manipulating the viral genome. We have developed methods for site-specific insertion and deletion mutagenesis of CMV utilizing a modified Escherichia coli lacZ gene as a genetic marker. The lacZ gene was placed under the control of the major beta gene regulatory signals and insert...

cloning, expression and library construction for hiv-1 tat protein

background: designing novel therapeutic agents has been a critical challenge for hiv disease. materials and methods: in current study a dna sequence which was encoded the tat protein was synthesized and inserted in pet 28 vector. vector was cloned in bl21-de3 e. coli and cultured in tb media. after protein expression, recombinant tat protein was purified by nta affinity chromatography. the tat ...